Financial support: The
authors gratefully acknowledge financial support from the National Science
Foundation under a grant awarded to L.Z. Santiago-Vázquez (awards #0310283 and
0514500) and the Center of Excellence in Biomedical and Marine Biotechnology,
Florida Atlantic University. R.G. Kerr acknowledges
support from theNatural Sciences and Engineering Council of Canada
(NSERC), the Canada Research Chair Program and the Jeanne and Jean-Louis
Lévesque Foundation.

While there is a
significant and growing body of knowledge describing the microbial communities
of marine invertebrates such as sponges, there are very few such studies
focused on octocorals. The octocoral Eunicea fusca is common on reefs in
various regions of the Caribbean and has been the subject of natural product
investigations. As part of an effort to describe the microbial community
associated with octocorals, a culture-independent analysis of the bacterial
community of E. fusca was conducted. Specifically, a 16S rDNA clone
library analysis was performed to provide baseline data. A total of 40 bacteria
members from 11 groups were found. In general, Proteobacteria were the dominant
group with a total of 24 species and α-Proteobacteria represented the
highest percentage of bacteria associated with E. fusca (27.5%). Other
prominent groups observed were Acidobacteria, Actinobacteria, Cyanobacteria,
Planctomycetes, δ-Proteobacteria, Lentisphaerae and Nitrospirae. This is
the first analysis of bacterial populations associated with the gorgonian E.
fusca.

Introduction

The octocoral Eunicea
fusca (Subclass Octocorallia, Order Gorgonacea, Family Plexauridae) is the
source of anti-inflammatory compounds such as fuscol and the fuscosides A and
B. These act as selective inhibitors of leukotriene synthesis (Shin and Fenical, 1991; Jacobson and Jacobs, 1992a; Jacobson
and Jacobs, 1992b). As
exemplified by E. fusca, a variety of structurally unique, bioactive
compounds have been isolated from octocorals (Faulkner,
2002). It has been suggested that bacteria living in symbiosis
with these organisms have an important role in the production of these
bioactive compounds (Radjasa and
Sabdono, 2009). For instance, a Vibrio sp. isolated from the surface of the soft coral Sinularia polydactyla is
the producer of Aqabamycins A-G, novel metabolites with antibacterial and
cytotoxic activities (Al-Zereini et al.
2010). Even though octocorals are well known
for their secondary metabolites, their associated microbial communities are relatively
understudied in comparison to other well-studied marine invertebrates such as
sponges and scleractinian corals (Enticknap
et al. 2006; Littman et al. 2010).

In addition to their
possible involvement in secondary metabolite production, symbiotic bacteria may
also contribute to the coral’s physiology and health (Rosenberg
et al. 2007; Shnit-Orland and Kushmaro, 2009). Since corals have no adaptive immune system, bacteria
associated with healthy corals are believed to play an important role in innate
immunity (Reshef et al. 2006). For instance, it
has been hypothesized that the microbial community of the mucopolysaccharide
surface layer of Gorgonia ventalina plays a role in protection against
disease by occupying niches that otherwise might be inhabited by pathogens, and
also by producing antimicrobial compounds (Gil-Agudelo
et al. 2006). Bacterial biofilms have been
implicated in promoting larvae metamorphosis of the scleractinian corals (Webster et al. 2004).
It is possible that some of these bacteria can enter into a symbiotic
relationship with the developing coral. Some of the most exciting and recent
studies are examining the relationship between coral disease and bacterial
population changes. For instance, microbial communities associated with Acropora
millepora were shown to change during bleaching events shifting to Vibrio-dominated
communities just prior to visual bleaching signs (Bourne et al. 2008).
Changes in bacterial populations associated with bleached or azooxanthellate
corals (Koren and Rosenberg, 2006) and with diseased corals have also been reported (Sunagawa et al. 2009).
Therefore, an understanding of microbial communities in the relatively
unstudied octocorals is of great importance.

The small number of
studies that have examined the microbial assemblages of soft corals have
reported a predominance of γ-Proteobacteria (Brück
et al. 2007; Santiago-Vázquez et al. 2007). Studies that have examined the culturable bacteria of
soft corals have also observed an abundance of γ-Proteobacteria followed
closely by α-Proteobacteria (Ivanova
et al. 2005) in addition to a minor presence
of Bacteroidetes in the soft coral Paragorgia arborea (Nedashkovskaya et al. 2005). Interestingly, an examination of bacteria associated with Plexaura fusifera showed that unbleached corals were dominated by low
G+C Firmicutes whereas bleached corals were dominated by γ-Proteobacteria
demonstrating the specificity of these relationships (Frenz-Ross et al. 2008). While little is known about bacterial communities in octocorals,
scleractinian coral-bacteria associations have been found to be specific (Rohwer et al. 2002; Lema et al. 2012).

The goal of this study is
to examine the bacterial communities closely associated with E. fusca by culture-independent techniques (CIT; 16S rDNA clone library) to provide a
baseline for other studies. CIT approaches are known to provide a broader
analysis of bacterial populations when compared to culture dependent studies.
It has been estimated that only 0.1% to 1% of microorganisms can be cultured in
the laboratory (Jannasch and Jones, 1959). The DNA of the bacteria was amplified by universal
eukaryotic 16S rDNA primers and a clone library was constructed. Restriction
fragment length polymorphism (RFLP) analysis was used as a dereplication tool. Subsequently,
PCR amplicons of interest were sequenced and analyzed. In accordance with
previous observations from other soft corals, it was shown that
γ-Proteobacteria were the most prominent bacteria observed. A study of the
microbial ecology of this coral will contribute to the identification of
potential producers of secondary metabolites and will provide a preliminary
baseline of the bacterial community of the coral in its healthy state.

Eunicea fusca was collected at Hillsboro Ledge, Deerfield Beach, Florida, USA,
on April 2005 by SCUBA diving at depths of approximately 10 m. Coral colonies
were handled with gloves and stored in individual plastic bags. Individual
colonies were rinsed several times with sterile seawater, immediately flash
frozen in liquid nitrogen, and stored at -80ºC until DNA extraction was
performed.

DNA extraction

Genomic DNA (gDNA) was
extracted from 100 mg of flash frozen E. fusca using the DNeasy Plant
Mini Kit (Qiagen, Valencia, CA, USA). E. fusca was ground with liquid nitrogen
using a sterile mortar and pestle. The coral slurry was transferred to a
sterile 1.5 mL centrifuge tube and DNA was extracted according to
manufacturer’s instructions. Samples were eluted 2X in a total of 60 µL of
ddH2O (double distilled water). Extracted DNA was analyzed by gel
electrophoresis. Samples were loaded, along with the appropriate molecular
weight markers, on a 1.5% (w/v) agarose gel, 1X TBE (Tris-Borate EDTA) buffer,
pH 8.3 and 1 µg/µL ethidium bromide. Electrophoresis was performed at 100V for
60 min. DNA bands were visualized in a Typhoon 9410 Imager (GE Healthcare,
Piscataway, NJ, USA). DNA concentration was quantified by reading the
absorbance at 260 nm on a SmartSpec 3000 spectrophotometer (Bio-Rad, Hercules,
CA, USA).

Amplification and purification
of 16S rDNA

Purified DNA from coral
tissue was diluted to 1:10, 1:100, and 1:1000 of the original concentration
with ddH2O. These dilutions were used for 16S rDNA amplification with universal
eubacterial primers FC27 (5’- AGAGTTTGATCCTGGCTCAG-3’) and RC1492 (5’TACGGYTACCTTGTTACGACTT3’;
Mincer et al. 2004). Each 50 µL PCR
reaction consisted of 1 µL template DNA at the different concentrations, 25 µL
Go Taq Green Master Mix (Promega, Valencia, CA, USA), 1 µL of each primer, and
22 µL of ddH2O. PCR cycling conditions were as follows: initial denaturation at
95ºC for 5 min, followed by 35 cycles of 95ºC for 30 sec, 55ºC for 30 sec, 72ºC
for 1.5 min. A final extension of 7 min at 72ºC was added. PCR products were
confirmed by gel electrophoresis. PCR amplicons of the anticipated size
(~1.5kb) were selected for cloning. The PCR products were eluted from the gel
with the MinElute Gel Extraction Kit (Qiagen) following manufacturer’s
instructions. Samples were eluted twice in a total of approximately 10 µL. Two
μL of the eluted product was loaded on a 1.2% agarose gel alongside a
quantifying ladder 1 kb Plus DNA Ladder E-Gel® DNA markers (Invitrogen,
Carlsbad, CA) to estimate quantity of the eluted PCR amplicon in preparation
for cloning.

16S rDNA cloning

The 16S rDNA fragments
were cloned using the TOPO TA Cloning Kit for Sequencing according to the
manufacturer’s instructions (Invitrogen, Life Technologies, Carlsbad, CA, USA).
A 100 μL aliquot from each transformation was plated onto pre-warmed
selective LB plates containing Ampicillin (50 mg/mL) and incubated over night
at 37ºC. A minimum of 96 clones from each cloning plate were randomly selected
and PCR-amplified using M13F (GTAAAACGACGGCCAG) and M13R (GTAAAACGACGGCCAGT)
primers following manufacturer’s cycling conditions. PCR products were
separated by 1.5% (w/v) agarose gel electrophoresis and visualized on a
Typhoon. Clones with the correct amplicon showed a single band at approximately
1700 bp. The number of clones ultimately picked was determined by the
subsequent diversity observed by RFLP.

The 16S rDNA sequences
were subjected to Basic Local Alignment Search Tool (BLAST) analysis at the
National Center for Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov).
The nucleotide sequences of the 16S rDNA gene sequences reported are available
in GenBank under accession numbers EF657844 to EF657883.

Phylogenetic analysis

The 16S rDNA sequences of
the clones were aligned and a using MegAlign (Lasergene) and the ClustalW
algorithm. Subsequently, a phylogenetic tree of the 16S rDNA sequences was
constructed using Phylip and the neighbour-joining method. Each reconstructed
group was statistically evaluated by bootstrapping with a 1000 replicates (Nei and Kumar, 2000).

Results and Discussion

This study analyzed the
microbial community of bacteria associated with Eunicea fusca. There is
evidence demonstrating that bacteria may be the source of biologically active
natural products initially isolated from octocorals and in other marine
organisms (Unson et al. 1994; Radjasa and Sabdono, 2009; Al-Zereini et al. 2010; Donia
et al. 2011) and thus it has become a priority to gain a detailed
understanding of the microbial communities in such systems. This knowledge may
lead to an efficient and sustainable supply of these compounds that does not
rely on harvesting coral tissue from the environment. In addition, the
physiological role of these bacteria in the octocoral host is not understood.

A 16S rDNA clone library
was constructed from an E. fusca genomic DNA sample. The resulting
transformants were screened by RFLP analysis with HhaI as the
restriction endonucleases that allowed for the clustering of similar clones. This
analysis analyzed a total of 310 clones with the presence of 40 unique OTUs. Representatives
of these clones were chosen for sequence analysis.

A survey of the identified
taxa revealed candidates that hold promise as potential producers of bioactive
secondary metabolites. These include the γ-Proteobacteria EF0605 (Accession
no. EF657879). Evidence suggests that the related marine bacterium Pseudomonas
rhizosphaerae may be the producer of potent antibacterial and antilarval
secondary metabolites (Qi et al. 2009). Similarly, Pseudomonas spp. isolated from marine
sources were found to be the source of potent antimicrobial compounds (Needham
et al. 1994; Charyulu et al. 2009). A second
candidate is the γ-Proteobacteria EF1505 (EF657861). This bacterium is
related to Candidatus Endobugula sertula, the endosymbiont bacterium of
the bryozoan Bugula neritina and also the known source of the
bryostatins, a family of macrocyclic lactones with anticancer activity (Davidson et al. 2001).
Finally, EF0705 (EF657868) and EF3305 (EF657846) showed a close phylogenetic
association to actinobacteria. Marine actinomycetes are well known prolific
sources of secondary metabolites (Lam,
2006).

Coral bacteria are
believed to play an important role in the host’s health. Corals are known to
have symbiotic relationships with nitrogen-fixing bacteria (diazotrophs; Lema et al. 2012) therefore
bacteria were examined for their potential involvement in the nitrogen cycle. A
group of five α-Proteobacteria, EF2205 (EF657848), EF2405 (EF657850),
EF2505 (EF657870), EF2605 (EF657871), and EF3905 (EF657852) were found to be
related to Pseudovibrio denitrificans (AY486423). This coastal seawater
organism is a facultative anaerobe capable of denitrification (Shieh et al. 2004).
This process may allow bacteria to contribute to the health of the host by
removing excess nitrate, and therefore controlling the population of the
symbiotic dinoflagellate Symbiodinium. Excess Symbiodinium and
the subsequent production of harmful reactive oxygen species (ROS) are
contributing factors to coral bleaching (Downs
et al. 2002; Merle et al. 2007). Another isolate potentially involved in the Nitrogen
cycle of E. fusca is the Nitrospirae EF0705 (EF657868). This bacterium
is related to Nitrospira marina, a chemolithotrophic nitrite-oxidizing
bacterium (Watson et al. 1986). In addition to nitrogen, bacteria involved in the carbon
cycle are known to be associated with corals (Wegley et al. 2007). The species EF0505
(EF657862), part of the CFB group, is closely related to CFB found in coastal
ocean bacterioplankton known to express transporter genes in response to DOC
and therefore contribute to carbon turnover (Poretsky et al. 2010). Excess
dissolved organic carbon (DOC) is detrimental to the health of corals (Kline et al. 2006).

Our findings concur with
data reported for other octocorals (Brück et al. 2007; Santiago-Vázquez et al. 2007) and suggest that a more in depth assessment using other
techniques such as fluorescent in situ hybridization (FISH) and next
generation sequencing is warranted. Such tools can be used to examine how these
associations vary with geographical location and health state of E. fusca.

It can be concluded from
this initial study that E. fusca hosts a diverse and complex assemblage
of coral-associated bacteria dominated by Proteobacteria, many of which are
only distantly related to previously described bacteria. This is the first
study of the microbial community associated with the coral E. fusca which provides a baseline for further studies in the areas of coral health and
natural products.

Acknowledgments

The authors acknowledge
the crew of the MV Diversity (Florida, USA) for assistance with the
collections, and Julie J. Enticknap for her assistance with phylogenetic data
analysis.